The autoantibody response in systemic autoimmune diseases focuses on a specific set of autoantigens, but it is not clear why. Via collaboration, the Rothstein and Shlomchik labs previously demonstrated that activation of rheumatoid factor (RF)-specific B cells by their antigenic immune-complexes was greatly potentiated if chromatin was contained in these complexes, and this was dependent on intact Toll-like receptor (TLR) signaling, most likely via TLR9. These in vitro findings led to the central hypothesis that certain autoantigens (particularly chromatin) can be ligands for TLRs or other innate immune response receptors, and that the ligation of such receptors can play a critical role in autoimmune responses of B cells. More recently we demonstrated that indeed TLRs regulate autoimmunity in vivo, a concept also corroborated by others in several systems. Specifically, we showed that TLR9 was required for anti-DNA type ANAs and that RNA-associated autoantibodies required TLR7. Together these discoveries have led to a new understanding of the pathogenesis of autoimmune diseases, as well as treatment approaches, some of which is already reaching the clinic. Though we found that presence of TLR7 (and MyD88) promoted disease, we unexpectedly found that TLR9 paradoxically suppressed global disease. These findings raise two major questions: a) how does TLR9 play a regulatory role in disease; and b) are TLR7 and TLR9 the only innate-immune sensing receptors of relevance in systemic autoimmunity? We will address these questions using in vivo genetic and in vitro biochemical approaches. In Aim 1 we will test the hypothesis that TLRs have tissue-specific functions in vivo, in part explaining the effect of deletion of TLR9 in all tissues. To do so, we will create and analyze floxed alleles of TLR7 and 9, as well as utilize an existing floxed MyD88 allele. These will be crossed with tissue-specific Cre alleles that we are maintaining on the MRL/lpr background. In Aim 2 we will test the hypothesis that TLR9 overexpression-either globally, or in specific cell types-will suppress some or all aspects of disease, thus highlighting a therapeutic approach. Again, we will use genetic approaches to create novel overexpressing mice. In Aim 3 we will use F2 genetic crosses to test the roles in disease of the remaining known innate immune nucleic acid sensing pathways and the inflammasome pathway. Aim 4 will directly evaluate the mechanism by which TLR9 suppresses disease. It will use in vitro biochemical and in vivo approaches to test several hypotheses concerning interactions between TLR9 and TLR7, as our unpublished genetic evidence shows that disease exacerbation due to TLR9 deletion requires TLR7. These studies should provide novel insights into how innate immune signaling regulates systemic autoimmune disease in vivo. In addition, we will generate and characterize valuable new reagents for the study of innate immunity in autoimmune disease and in general.